1. Field of the Invention
The present invention relates to a biochip and a manufacturing method thereof. More particularly, the present invention relates to a micro-array biochip with dot array patterned reagents and a manufacturing method thereof, and especially to a method for forming a biochip with a bonding barrier layer by vapor deposition.
2. Description of the Related Art
The term microarray biochip refers to a substrate with a plurality of various reagents immobilized on the surface. Suitable materials of the substrate can be glass, silicon chip, nylon film, or polymer. These reagents (also called probes) are usually designed according to the specificity or other active characteristics, for example, binding affinity, being complementary to biological samples (i.e. subjects), and so on. Under actual experimental conditions, a biological sample is added to the substrate on which probes are fixed, and simultaneously the probes on the substrate react with the subjects in the biological sample by hybridization. By detection and analysis using various marking systems and specific instruments, information on the quality and quantity of the subjects related to the tested biological sample is obtained.
In the conventional biochip technique, a commonly used bonding barrier material is bioserum albumin, such as horse serum albumin and bovine serum albumin, which is used to block various binding of the excess biomedical molecules and the surface at the non-detected locations, so as to reduce background noise, and thereby achieve the purpose of enhancing the detection sensitivity. Taking fluoroimmunoassay as an example, when bioserum albumin is used as the bonding barrier in the fluoroimmunoassay, the bioserum albumin is added as a bonding barrier layer on the chip with a plurality of first biomedical molecular dots arranged thereon. Therefore, subsequent biomedical molecules subsequently overlaid on the biochip are prevented from binding to the surfaces of the biochip without the first layer of biomedical molecules through non-specific bonding (binding for non-specific identification), such that they serve as the barrier layer to reduce the effect of the background fluorescence, thereby improving the S/N ratio of the fluorescence detection.
FIG. 1(a) is a diagram showing a fluorescence scanning image after a bonding barrier layer of bioserum albumin is formed on a biochip through liquid soakage. The black shown in the background confirms that the bioserum albumin effectively eliminates the background value caused by the non-specific fluorescence signals. However, since the overlaid bonding barrier layer is formed by liquid soakage, the previous first layer of biomedical molecular dots will be dispersed by the liquid. Therefore, a part of the background noise is raised, and the signal points are blurred and dispersed. Additionally, in the detection step, in order to rinse the excess and non-bonded protein, it is necessary to use a buffer solution with a surfactant to rinse the biochip, so that the tailing phenomenon will easily occur at the moment of rinsing with liquid, and other signal points will be contaminated. Therefore, although the biochip with bioserum albumin as bonding barrier has a higher S/N ratio than the biochip without bonding barrier, the biochip with bonding barrier will present an inferior fluorescence scanning image due to the tailing or blurring, as shown in FIG. 1(a).
Furthermore, the conventional bioserum albumin is easily attached on the surface of the first layer of biomedical molecules, so that the ability of the second layer of biomedical molecules to identify the first biomedical molecular layer is blocked. Thus, the practical value of the detection signal is less than expected. Therefore, by comparing the results of fluorescence signals in FIG. 1(a) (in which a bioserum albumin is used as bonding barrier material) and those in FIG. 1(b) (which use no bonding barrier material), it can be clearly observed that the green and red fluorescence signals at the round points are low (the overlap of red and green fluorescence produces yellow). That is to say, the bioserum albumin bonding barrier material and the first biomedical molecular layer are adhered to each other, so that the green fluorescence signal for labeling the first biomedical molecular layer is reduced. Consequently, the undesired adherence as shown in FIG. 1(a) also reduces the identification binding rate of the second biomedical molecular layer, so that the red fluorescence signal for labeling the second biomedical molecular layer becomes low in comparison with the first biomedical molecular layer without bonding barrier (FIG. 1(b)).
FIGS. 2(a)-2(i) are diagrams of respective steps for forming a biochip with a bonding barrier of bioserum albumin through liquid soakage. As shown in FIGS. 2(a) and 2(b), a cleaned substrate 21 or a slide is soaked in a liquid to form a self-assembled monolayer 22 thereon, for example, a layer of (3-aminopropyl)trimethoxysilane (APTS). Then, an excess first biomedical molecular layer 23 is dropped on the substrate 21 by a micropipette (as shown in FIG. 2(c)). After the first biomedical molecular layer 23 and the self-assembled monolayer 22 on the substrate 21 are sufficiently combined through bonding reaction, the excess portion of the first biomedical molecular layer 23 is rinsed out by a liquid 26, but the tailing phenomenon occurs in each point of the first biomedical molecular layer 23, as shown in FIG. 2(d). A bioserum albumin 24 capable of being a bonding barrier is then dropped (as shown in FIG. 2(e)), and the bioserum albumin 24′ is uniformly distributed on the surface of the self-assembled monolayer 22 with a cover glass. The portion of the self-assembled monolayer 22 without the first biomedical molecular layer 23 dropped thereon is subjected to bonding barrier processing, as shown in FIG. 2(f). Referring to FIG. 2(g), the excess bioserum albumin 24″ serving as a bonding barrier is rinsed away with a liquid 27. Since the adherence still exists between a part of the bioserum albumin and the first biomedical molecular layer 23 after the aforesaid rinse (as shown in FIG. 2(h)), the identification rate is reduced when the first biomedical molecular layer 23 is identified by a second biomedical molecular layer 25 (as shown in FIG. 2(i)) and the second biomedical molecular layer 25 is blocked by the bioserum albumin 24″ which is adhered to the surface of the first biomedical molecular layer 23.
In view of the foregoing, there is a need for a method to improve the bonding barrier effect in manufacturing the biochip, so as to avoid various disadvantages resulting from the conventional bioserum, to further improve the detection efficacy and shorten the manufacturing process time, and to benefit from the advantages of batch processing.